Janelle A. Noble, Ph.D. has a very active research program in the genetics of susceptibility to complex disease, with a major focus on type 1 diabetes. Dr. Noble’s group has studied the HLA region genes for the past 16 years, and performs HLA genotyping assays both for their research projects and in collaboration with other investigators outside CHORI. The HLA genes are the most polymorphic in the human genome; for example, the HLA-B gene has over 1000 different variants (alleles) reported to date. Although the major focus of the research program is type 1 diabetes, she has active research collaborations involving other autoimmune diseases, including lupus, MS and rheumatoid arthritis. Past and future collaborative projects include topics as diverse as the genetic basis of the tendency to stroke in children with sickle cell disease, association of HLA genes with age-related macular degeneration, and ethnic health disparities in the prevalence of bacterial vaginosis.

1: The genetics of type 1 diabetes autoimmunity

HLA disease association. This study has been ongoing since 1993, with a clinical component that began in 1996. The goal of this study is to look for genetic associations with type 1 diabetes (T1D) both in existing collections and in newly-collected samples from Children’s Hospital and Research Center Oakland (CHRCO). The T1D studies of both HLA and other candidate loci on existing collections, such as the T1D families from the Human Biological Data Interchange (HBDI), have resulted in numerous publications (see, e.g. (Noble et al., 2002; Noble et al., 1996; Noble et al., 2006; Noble et al., 2000; Noble et al., 2003; Valdes et al., 2005). Collaborative studies on samples from cohorts assembled by other investigators have also led to new insights about the genetic basis of T1D susceptibility (see, e.g., (Cruz et al., 2004; Noble et al., 2008). Collection of samples from CHRCO began in 1996, and the current collection includes samples from more than 130 diabetes patients, with a very diverse mix of ethnicity, and nearly 200 relatives of those patients. The current focus of the T1D work in the Noble lab is on African American samples. HLA alleles and haplotype combinations differ strikingly between African-derived and European-derived chromosomes. Recently, the combination of data from the African American subjects collected at CHRCO and from the Type 1 Diabetes Genetics Consortium (see below) has enabled statistical analysis for T1D association of African American patients vs. population-based African American HLA data, revealing novel, and sometimes unexpected, T1D susceptibility effects for African HLA alleles and haplotypes. For the population-based data, DNA was prepared from 1000 newborn bloodspot cards from self-reported African Americans used with permission from the State of California Genetic Disease Branch. HLA DRB1, DQA1, and DQB1 have been genotyped; genotyping for the remaining classical HLA loci, including DPA1, DPB1, A, B, and C is ongoing. African alleles are severely understudied and may contain previously undetected polymorphisms. We plan to use a sequence-based technology to elucidate previously-unreported polymorphisms.

Admixture analysis. Recently, a subset of the population-based African American samples, a subset of the African American T1D patients, and a small number of non-diabetic African American controls were assessed for genetic ancestry using a panel of 106 ancestry informative markers (AIMs). African Americans represent a mix (admixture) of African-derived genes and European-devived genes, sometimes with some Native American genes as well. Even with the very small sample numbers, the data showed global differences in admixture proportions among the three groups, with a statistically significant difference between the patients and the non-diabetic controls (unpublished data). In addition, the admixture data on the population-based samples reveals the presence of outliers (samples reported as African American but exhibiting 100% Caucasian ancestry) in the collection. These data have prompted the planned study of a much larger set of AIMs (~1500) to generate accurate ancestry estimates for this group. These preliminary data, showing differences in admixture proportions between African American T1D patients and controls, underscore the need for additional, comprehensive admixture analysis of these samples to reveal novel disease susceptibility regions. We will then perform fine mapping and resequencing of novel disease susceptibility regions.

Association of non-HLA loci with T1D. Over the years, a number of non-HLA candidate T1D susceptibility gene loci have been reported by many groups, including Dr. Noble’s (Ladner et al., 2005; Mirel et al., 2002; Noble et al., 2003). The effects of most of these genes on T1D is modest, particularly when compared to that of HLA. In some cases, the associated polymorphisms may be causal, but in many cases, they simply mark a predisposing or protective haplotype. In addition, most studies focus on only one or a very few candidate loci at a time. In many cases, these studies produced conflicting results, perhaps due to differences in the small cohorts in which they were tested. Recently, the T1DGC (www.t1dgc.org; see below) has completed a “rapid response” project in which associations of candidate loci, reported from small studies, were tested in thousands of samples collected for the T1DGC project. Dr. Noble is exploring new technology to facilitate the ability to easily test all T1D genetic markers rapidly on large cohorts of samples. This is likely to reveal gene-gene interactions and may lead to candidate marker sets for development of diagnostic tests.

Biological follow-up of genetic associations. Dr. Noble is following up her initial finding of T1D association with a polymorphism in the TCF7 gene (Noble et al., 2003) with planned biological studies. The initial association data have been replicated in two large cohorts, including the T1DGC. TCF7 is a T cell specific transcription factor that is hypothesized to play a role in the lineage fate of T cells and, thus, is implicated in autoimmunity. Planned experiments include analysis of differences in transcript fingerprints between the major (T1D neutral) and minor (T1D susceptible) alleles of the TCF7 gene. Both in vitro cell culture experiments and, in collaboration with colleagues at USCF, in vivo mouse models of diabetes will be tested. As an alternative to the usual microarray analysis for mRNA expression levels, these experiments are being planned using the new “digital gene expression” technology, in which cDNA from expression studies is directly sequenced on a massively parallel sequencing platform, such as the 454, thus giving unequivocal and highly quantitative data reflecting transcript profiles. Using this system, differences in transcript profiles for different alleles of TCF7 can be measured and may lead to the identification of pathways with druggable targets for T1D intervention.

2: The Type 1 Diabetes Genetics Consortium (T1DGC) The T1DGC is an NIH-funded, international collaboration to collect and genotype thousands of T1D families, as well as patients and controls, in the search for T1D genetic susceptibility loci. Dr. Noble has been involved with the T1DGC since shortly after its inception in 2001, and she is the PI of the North American HLA Genotyping Center for the T1DGC effort. She was instrumental in developing the process that is used for the T1DGC HLA genotyping effort, which has resulted in nearly 100% concordance among the three T1DGC HLA genotyping centers (Oakland/Pleasanton, CA, Melbourne, Australia, and Malmö, Sweden) For concordance data, see www.t1dgc.org. At the inception of the project, the linear array methodology was selected as the most robust and cost-effective HLA genotyping method for the T1DGC effort. Protocols, instrumentation, reagents, and data interpretation rules were standardized for all laboratories. To date, more than 14,000 samples have been genotyped by the T1DGC HLA genotyping labs. The current T1DGC data set represents, arguably, the largest, most complete, and most accurate set of HLA genotyping data ever produced; however, the allele calls necessarily contain some ambiguity. Most of the ambiguity in the linear array system has no effect on the outcome of the association analyses. However, three sets of ambiguous allele pairs may have an effect on the data. Dr. Noble has recently proposed and been funded for a small ancillary study, in collaboration with Dr. Henry Erlich at Roche Molecular Systems, to utilize 454 massively-parallel sequencing technology to resolve these ambiguities. Samples are currently being selected and assembled from the collection, and the planned ambiguity analysis, involving over 350 of the T1DGC samples, will be completed with a single run of the 454 FLX instrument. Re-genotyping of the more than 14,000 samples from the T1DGC collection is neither cost-effective nor warranted at this time. However, the 454 technology will allow the in-depth analysis of subsets of the T1DGC data, e.g., the ~200 African American patients or other understudied groups. These experiments are currently in the planning stages. T1DGC HLA class II association data have been published for HLA DR and DQ-encoding genes (Erlich et al., 2008), and manuscripts for DPB1 and HLA class I results are in preparation.

3: Other Autoimmune Diseases: SLE, RA, MSDr. Noble in involved in a very active collaboration with Dr. Lisa Barcellos from UC Berkeley and Dr. Linsdey Criswell from UCSF, to study HLA association in autoimmune diseases in addition to T1D. Since 2007, the Noble laboratory has genotyped over 8000 samples for studies of systemic lupus erythematosis (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). Results from the SLE genotyping are currently being published, with one manuscript in press and another submitted for publication. In addition, Dr. Noble is the PI of the HLA genotyping subcontract for a program project for the SLE genetics consortium (SLEGEN), which is currently under review.

4. Other collaborative projects
In addition to the large projects described above, Dr. Noble has other active collaborations. These include an RA study with Dr. Laura Su from Dr. Mark Davis’ lab at Stanford, and HLA genotyping for the National Pancreatic Organ Donor network (nPOD).

5. Planned studies
Other proposed studies awaiting review and funding include a study of HLA association with age-related macular degeneration (AMD), in collaboration with Dr. Gregory Tranah of the California Pacific Medical Center Research Institute, and a study of the influence of HLA in bacterial vaginosis (BV), particularly in African American women, in collaboration with Dr. Jenifer Allsworth at Washington University. The BV study, with its emphasis on African Americans, is likely to reveal novel polymorphisms in HLA alleles that are previously undetected.

SUMMARY The Noble lab produces HLA genotyping data for multiple projects, ranging from supporting small, collaborative studies, to providing core laboratory services for large, international consortia. In addition, they study disease association of other candidate loci for T1D. Currently, they are planning experiments to follow up one of these associations with both in vitro and in vivo biological studies in an attempt to discover targets for disease intervention.